Spectrophotometric Determination of Protein by Bicinchoninic Acid Assay (BCA Method)

Overview of Sections

• Introduction
• Experimental
• Reagents, Instruments, and Materials
• Analytical Approach
• Analytical Quality Assurance
• Conclusion
• Related Products

Introduction

Proteins are large biomolecules and involved in many physiological processes within organisms. They consist of long chains of amino acids which are connected by peptide bonds. The quantification of proteins is an important field in bioanalytic. There are different methods of protein estimation in samples. One method is the BCA protein assay, which is based on the reduction of Cu²⁺ to Cu⁺ by proteins. Cu⁺ then reacts with BCA to form a purple-blue complex.

Experimental

This Application Note describes the BCA method for protein estimation. The method is preprogrammed on the corresponding Spectroquant^® Prove plus photometers with firmware version 1.5 or above, so that the calibration that needs to be done for each assay, can be performed directly on the instrument itself. All reagents required for the measurement are included in the test kit.

More details can be found in the packaging insert of the test kit.

BCA Method Principle

In alkaline medium proteins reduce Cu²⁺ to Cu⁺, which then reacts with bicinchoninic acid (BCA) to form a purple-blue complex that can be measured photometrically at 562 nm.

Measuring range

• 200–1000 μg/mL protein as bovine serum albumin (method “Protein BCA”, No. 319)

Sample material

• Aqueous samples or samples after appropriate sample preparation.

Reagents, Instruments, and Materials

Protein Assay Kit

• Bicinchoninic Acid Protein Assay Kit (BCA1-1KT)
• Albumin fraction V (from bovine serum) (112018) (Optional, if calibration is performed)

Instruments

For the protein measurement, one of the following Spectroquant^® photometers is necessary:

• Spectroquant^® UV/VIS Spectrophotometer Prove 600 plus (1.73028)
• Spectroquant^® UV/VIS Spectrophotometer Prove 300 plus (1.73027)
• Spectroquant^® VIS Spectrophotometer Prove 100 plus (1.73026)

Note: Legacy instruments Prove 100/300/600 are also suitable.

Software for data maintenance

The Spectroquant^® Prove Connect to LIMS software package provides an easy way to transfer your data into a preexisting LIMS system. This software can be purchased under:

• Prove Connect to LIMS (Y11086)

Materials

• Rectangular cells 10 mm (glass) (1.14946) or
• Semi-micro rectangular cell 10 mm (glass)*(Z600288) or
• Rectangular cells 10 mm (polystyrene)*(C5291)
• Flat-bottomed tubes with screw caps (1.14902)

*Due to the automatic cell detection of the Prove instruments it is important to use cells or semi-micro cells with complete side walls.

Only necessary for turbid solutions:

• Filter or centrifuge

Analytical Approach

Sample preparation

• The sample solution should be clear. Centrifuge or filter turbid solutions.
• If the concentration of protein is higher than 1000 μg/mL, the sample should be diluted with the sample solvent so that measurement can be carried out within the measuring range.

Preparation of calibration solution

It is necessary to create a standard curve for each assay. Therefore, prepare the stock- and standard solution as follows: For the stock solutions dissolve precisely 100 mg of Albumin fraction V (BSA) in 100 mL of buffer used as sample solvent in a volumetric flask. Distilled water can also be used but interferences originating from the buffer may not be compensated by using water. The BSA-concentration of this solution is 1 μg/mL. Use the stock solution to prepare the standard solutions used for calibration, see following table:

	Protein Concentration (µg/mL)
	0	200	400	600	800	1000
1 µg/mL BSA Protein stock solution I (mL)	-	0.20	0.40	0.60	0.80	1.00
Sample buffer (recommended) / water (mL)	1.00	0.80	0.60	0.40	0.20	-
The standard solutions can be aliquoted and stored for approx. 6 months at -20 °C.

Preparation of Measurement Solutions

• For details on how to prepare the measurement solutions see packaging insert of the test kit “BCA1-1KT”.¹ Prepare the sample, reagent blank and standard solutions according to the procedure for the test tube assay step 1-6 (see packaging insert “A. Standard 2.1 mL Assay Protocol”).

Measurement

• Open the method list (<Methods>) and select method No. 319 “Protein BCA”.
• For each measurement series, a zero adjustment is required. It is recommended to use the same cell for zero adjustment and for sample measurement. The zeroing procedure for the measurement series is automatically prompted by the instrument. For zero adjustment fill the 10-mm rectangular cell with the solvent used for dilution. After prompting, place the filled rectangular cell in the cell compartment, the zero adjustment is executed automatically. Confirm the implementation of zero adjustment with "OK". The zero adjustment is valid for the entire measurement series.
• A user-defined calibration is necessary for each assay. Do this by tapping the <Settings> button and selecting the <RECALIBRATION> menu item. An input mask pops up.
• Tap twice on <+> in the numerical keyboard to create two additional input lines.
• Select the “Absorbance” field in the “E0” line (selected fields are shown in a blue frame).
• Fill calibration solution E0 into a 10-mm rectangular cell and insert cell into the cell compartment. The measurement starts automatically. The measured absorbance is shown in the display.
• Select the “Conc.” field in the “1” line and enter the concentration of 200 μg/mL for the first calibration solution.
• Select the “Absorbance” field in the “1” line. Fill calibration solution 1 into a 10-mm rectangular cell and insert cell into the cell compartment. The measurement starts automatically. The measured absorbance is shown in the display.
• Select the “Conc.” field in the “2” line and enter the concentration of 400 μg/mL for the second calibration solution.
• Select the “Absorbance” field in the “2” line. Fill calibration solution 2 into a 10-mm rectangular cell and insert cell into the cell compartment. The measurement starts automatically. The measured absorbance is shown in the display.
• Select the “Conc.” field in the “3” line and enter the concentration of 600 μg/mL for the third calibration solution. Select the “Absorbance” field in the “3” line. Fill calibration solution 3 into a 10-mm rectangular cell and insert cell into the cell compartment. The measurement starts automatically. The measured absorbance is shown in the display.
• Select the “Conc.” field in the “4” line and enter the concentration of 800 μg/mL for the fourth calibration solution.
• Select the “Absorbance” field in the “4” line. Fill calibration solution 4 into a 10-mm rectangular cell and insert cell into the cell compartment. The measurement starts automatically. The measured absorbance is shown in the display.
• Select the “Conc.” field in the “5” line and enter the concentration of 1000 μg/mL for the fifth calibration solution.
• Select the “Absorbance” field in the “5” line. Fill calibration solution 5 into a 10-mm rectangular cell and insert cell into the cell compartment. The measurement starts automatically. The measured absorbance is shown in the display.
• Check linearity of the function by tapping on the graph symbol. Close the graph view by tapping <X> and activate the linear field, if the function is linear.
• Activate the <U-CAL on> fields.
• Optionally enter a batch number for the calibration, selecting the <Lot number> field to do so.
• Once all calibration solutions have been measured, save the calibration by pressing <OK>.
• If a dilution step for the samples has been performed, enter the factor. Therefore, tap the <Settings> button and select the <DILUTION> menu item. Enter the dilution factor in the form 1+x.
• Measure the measurement sample. Therefore, fill the measurement sample into a 10-mm rectangular plastic cell and insert the cell into the cell compartment. The measurement starts automatically.
• Read off the result in μg/mL from the display.

Data transfer from Prove spectrophotometers (optional)

After measurement, transfer the values measured on the Prove spectrophotometer using the software “Prove Connect to LIMS”.

Influences of Foreign Substances

For details on interfering substances, see the Compatibility Chart in the Product Information Sheet - BCA1 ¹ of the test.

Analytical Quality Assurance

The objective of analytical quality assurance (AQA) is to secure correct and precise measurement results.

AQA is recommended before each measurement series. To check the measurement system (test reagents, measurement device, and handling) a self-prepared bovine serum albumin standard solution can be used. For details on how to prepare the standards see calibration-preparing the standard solutions. Sample-dependent interferences (matrix effects) can be determined by means of standard addition.

For details on how to perform the AQA check see the instrument-specific manuals.

Conclusion

The Protein (BCA) assay kit is an easy and fast way to quantify protein concentration in your sample. The measurement can be performed without high-cost instrumentation.

REFERENCES

1. Product Information Bicinchoninic Acid Kit for Protein Determination, Sigma-Aldrich, Cat. No. BCA1 from 05/2012. [Internet]. Available from: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/256/958/bca1pis-mk.pdf